Integrase (IN) is encoded by the Pol gene from the HIV provirus. Our laboratory can efficiently express IN as an active recombinant protein, and has pioneered the integrase inhibitors research field (PNAS 1993), discovered several families of lead inhibitors, demonstrated that IN inhibitors act as interfacial inhibitors (Nature Rev Drug Discovery 2012), and been granted several patents for IN inhibitors for therapeutic development. Our current studies are focused on the optimization of our novel chemotype integrase inhibitors to overcome resistance to raltegravir, elvitegravir and dolutegravir and target novel sites of IN. We have published and patented novel synthetic chemotypes as IN strand transfer inhibitors (INSTIs) including phtalimide and quinolinonyl derivatives in collaborations with Dr. Terrence Burke, Laboratory of Medicinal Chemistry (CCR, NCI). We have developed a panel of recombinant IN proteins bearing the mutations observed in patients that develop resistance to raltegravir, elvitegravir and dolutegravir. Using our resistant IN mutants, we have characterized the molecular pharmacology of elvitegravir, dolutegravir and our novel inhibitors, comparing them to raltegravir. We have shown that raltegravir, elvitegravir, dolutegravir and our novel series are highly selective for the strand transfer reaction, while being more than 100-fold less potent against the 3'-processing reaction, and almost inactive against the disintegration reaction mediated by integrase. The selective activity against strand transfer (one of the 3 reactions mediated by integrase) demonstrates the very high specificity of the clinically developed IN strand transfer inhibitors (INSTIs). It is consistent with our pharmacological hypothesis (Nature Drug Discovery 2012) that the strand transfer inhibitors trap the IN-viral DNA complex by chelating the divalent metals in the enzyme catalytic site following 3'-processing of the viral DNA and with our co-crystal structure and molecular modeling data. We have characterized the biochemical enzymatic activities and drug sensitivities of the IN mutants that confer clinical drug resistance. We have expanded these studies to double-mutants in the integrase flexible loop that commonly arise in raltegravir-resistant patients. The working hypothesis is that the second mutation acts as gain of function to rescue the biochemical activity of IN after it had become defective by the presence of the first mutation. One of aims is to understand the molecular mechanisms of such complementation and the structural connections between the flexible loop, the viral and host DNAs, and the inhibitors. We found that the flexible loop double-mutant 140S-148H is cross-resistant to both raltegravir and elvitegravir but much less to dolutegravir and to some of our new derivatives. On the other hand, the 143Y mutant is primarily resistant to raltegravir and minimally resistant to elvitegravir and dolutegravir. These results provide a rationale for using elvitegravir in patients that develop resistance to raltegravir due to mutation 143Y (but not in the case of mutations 140S-148H). Our results support the value of dolutegravir to overcome resistance to raltegravir and elvitegravir and facilitate patient compliance. We have determined additional crystal structures of wild-type and mutant prototype foamy virus (PFV) intasomes bound to our new series of inhibitors in collaboration with Dr. Peter Cherepanov at the Crick Institute, Cancer UK Center in London. The ability to structurally adapt to the structural changes associated with drug resistance is now achievable to rationally develop our new INSTIs. This year, we have also performed biochemical experiments to dissect IN catalytic mechanism, especially the first step of its reaction, 3'-processing of the viral DNA end. Molecular modeling of HIV-1 integrase, together with biochemical data, indicate that the conserved residue Q146 in the flexible loop of HIV-1 IN is critical for productive viral DNA binding through specific contacts with the virus DNA ends in the 3'-processing and strand transfer reactions. Notably, we also showed the existence of a relationship between 3'-processing inhibition and the ability of INSTIs to overcome resistance to raltegravir, elvitegravir and dolutegravir. Our ongoing and proposed drug discovery effort will continue in collaboration with Terrence Burke at the NCI-CCR Chemical Biology Laboratory, Stephen Hughes at the NCI-CCR Drug Resistance Program, and Peter Cherepanov at the Francis Crick Institute in London. Because the XZ compounds possess high therapeutic index without cytotoxicity up to 250 microM (the highest dose tested), 3 of them, XZ419, XZ434 and XZ446 have been selected for preclinical development and animal testing by the NCI-CCR Drug Development Collaborative group (DDC). It is likely that further chemical modifications will be necessary to optimize formulation and pharmacokinetics. IATAP support will allow us to continue our collaborative work with Terrence R. Burke and XueZhi Zhao and with Stephen Hughes to achieve these goals.